A prion is an infectious protein, an altered form of a normal protein which has lost its normal function but has acquired the ability to convert the normal form of the protein into this same altered (prion) form. This concept arose in studies of scrapie, a CNS disease of sheep, and the same disease of man (kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann- Streussler-Scheinker (GSS) disease). We have proposed, based on genetic evidence, that the yeast non-Mendelian elements, [URE3] and [PSI] are prions, infectious altered forms of the cellular ure2p and sup35p, respectively [Wickner, R. B.: Science 264, 566-569 (1994)). Ure2p is a negative regulator of transcript ion of genes encoding enzymes for the catabolism of poor nitrogen sources. Sup35p is involved in the termination of translation. We find that overexpression of the entire Ure2p produces a 100-fold increase in the frequency with which [URE3] arises. Overexpression of just the N-terminal 65 residues of Ure2p is sufficient to induce [URE3] and the frequency is 8000-fold above the background level. Overexpression of the C-terminal part, lacking just the N-terminal 65 residues is sufficient to carry out the normal nitrogen catabolite repression activity of Ure2p, and this C-terminal domain cannot induce the prion change and cannot undergo the prion change itself. The earliest biochemical finding implicating the mammalian PrP in the scrapie phenomenon was that this protein was protease K-resistant in the brains of sheep with scrapie, but sensitive in normal sheep. We find that the Ure2p in extracts of [URE3] strains is much more protease X-resistant in comparison with wild-type [ure-o) strains. The fragments that are resistant to proteinase K digestion include the N-terminal prion-inducing domain.